1. Field of the Invention
The present invention relates to expansion of human hematopoietic stem cells.
2. Background
The hematopoietic stem cell (HSC) is the progenitor cell for all blood cells. It is through the proliferation and differentiation, gives rise to the entire hematopoietic system. HSCs are believed to be capable of self-renewal—expanding their own population of stem cells—and they are pluripotent—capable of differentiating into any cell in the hematopoietic system. From this rare cell population, the entire mature hematopoietic system, comprising lymphocytes (B and T cells of the immune system) and myeloid cells (erythrocytes, megakaryocytes, granulocytes and macrophages) is formed. The lymphoid lineage, comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides red blood cells, which act as oxygen carriers. As used herein, “stem cell” refers to hematopoietic stem cells and not stem cells of other cell types.
A schematic of hematopoiesis is shown in FIG. 8. This is a complex process which involves a hierarchy of HSC which can be influenced by a variety of external regulatory factors. Whether the fate of an individual HSC is determined by random or stochastic events or can actually be defined by external influences remains an important area of investigation. Morrison et al., Cell, 88:87-298, 1997; Krause, Oncogene, 21:3262-3269, 2002. To date attempts to create an in vitro environment which favors HSC self replication rather than commitment and differentiation has resulted in limited success Jiang et al., Oncogenes, 21:3295-3313, 2002; Guenahel et al., Exp. Hematol., 29:1465-1473, 2001; Srour, Blood, 96:1609-1612, 2000; Berardi et al., Science, 267:104-108, 1995; Heike et al., Biochim. Biophys. Acta, 1592:313-321, 2002.
Myelosuppression and myeloablation is often seen as a result of chemotherapy. Bone marrow transplantation, either autologous or allogeneic, can be used to replace a functional hematopoietic system. In addition, purified stem cells may be reinfused into the patient to restore hematopoiesis in these compromised patients. It also has been found that administration of chemotherapeutic agents and/or cytokines mobilizes bone marrow stem cells into the peripheral blood such that peripheral blood can be harvested as a source of stem cells. In an autologous transplant setting it is often particularly desirable to purify stem cells from the bone marrow or peripheral blood to use as a graft as a way of purifying long-term repopulating cells free of contaminating tumor cells. Tumor cells have been detected as high as 10% in mobilized peripheral blood collections and up to 80% in the mononuclear fraction from marrow.
Unlike whole bone marrow, stem cell replacement does not restore mature hematopoietic cells immediately. Due to the time necessary to generate mature cells from reinfused stem cells, there is a lag during which the patient remains immunocompromised. One proposed solution has been to expand the purified (and tumor-free) stem cells ex vivo to generate a cell population having both stem cells and slightly more differentiated cells, which would be able to provide both short- and long-term hematopoietic recovery.
Methods of bone marrow expansion have been developed, however, expansion of stem cells is not as straight-forward as expansion from a mature population. First, stem cells are very rare and, therefore, the number of stem cells isolated from any source will be very small. This reduces the size of the population that can be used to initiate the culture system. Second, the goal in stem cell expansion is not just to produce large quantities of mature cells, but also to retain stem cells and to produce many immature progenitor cells, which are capable of rapidly proliferating and replenishing mature cell types depleted in the patient. Upon reinfusion into a patient, the mature cells are cleared quickly whereas stem cells home to the marrow where long-term engraftment can occur (engraftment assays may be measured using for example, SCID mice using techniques well known to those of skill in the art). In addition, the immature progenitor cells can produce more cell types and more numbers of cells than the mature cells, thus providing short-term hematopoietic recovery. Stem cells are now regularly cultured on adherent monolayer of stromal cells, which supports the viability of stem and early progenitor cells (“Dexter culture”; see Dexter et al. (1976) J. Cell Phys. 9:335). For clinical use, it is preferable to utilize a more easily defined stromal-free culture system. U.S. Pat. No. 5,409,825 describes stroma-free stem cell expansion. U.S. Pat. Nos. 5,728,581, 5,665,557, 5,861,315, 5,997,860, 5,905,041, 6,326,198 each incorporated herein by reference in its entirety describe additional methods known to those of skill in the art for the expansion of stem cells in culture.
Despite the availability of numerous methods for in vitro expansion of HSCs in culture, these methods remain inadequate for the production of HSCs for transplantation that maintain self-renewal capacity and multipotency. This is may be due to these cells undergoing an epigenetically-mediated loss of gene function accompanied by DNA methylation of a gene's promoter and by histone deacetylation, thereby resulting in a loss of primitive HSC function. Although the molecular signature that defines an HSC has recently been described, the patterns of gene expression that lead to HSC self replication rather than commitment remain unknown (Santos et al., Science 298:597-600, 2002; Ivanova et al., Science, 298:601-604, 2002).
Primitive HSC are thought to maintain an open chromatin structure that permits access to the entire HSC developmental program while more differentiated cells along the hematopoietic hierarchy are thought to characteristically undergo a stepwise progression of epigenetic events that controls transcriptional events for each stage and class of progenitor cells (Akashi et al., Blood, 101:383-389, 2003). HSC promiscuously express a set of transcription factors that are restricted as the process of commitment to a particular pathway of differentiation occurs. Jiang et al., Oncogenes, 21:3295-3313, 2002; Akashi et al., Blood, 101:383-389, 2003; Hu et al., Genes Dev., 11:774-785, 1997). These events likely involve the activation and/or silencing of yet to be identified groups of pivotal genes that are influenced by a variety of epigenetic events.
In short, conditions previously utilized for in vitro stem cell expansion result in silencing of genes required for HSC to undergo symmetrical cell division. Accordingly, a need exists for methods and compositions for efficient culture and expansion of stem cells under controlled conditions that will yield suitable numbers of stem/progenitor cells for clinical use.